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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

• Information relating to the copy number of a target of interest in the genome of a biological sample may be desirable for a number of purposes, including basic research and clinical diagnosis of various diseases.
• One class of diseases where the copy number of a target of interest may be particularly desirable to know is cancer.
• Numerous cancers present with abnormal copy numbers of one or more genes. In many cases, there exists a positive correlation between copy number and the existence and/or progression of cancer. Therefore, determining the copy number of a target of interest in a sample from a patient of tissue suspected of being cancer tissue may be useful, for example, in diagnosing, treating, and/or monitoring the course of the patient' s cancer.
• qPCR quantitative polymerase chain reaction
• the technique when attempting to apply qPCR to determine the copy number of a target of interest in a cancer cell, the technique may be rendered difficult as a result of one or both of the random, evolving genomic abnormalities (e.g., aneuploidy) found in cancer cells and the modifications to nucleic acids that may occur when tissue samples are archived by formalin fixing and paraffin embedding (FFPE).
• FFPE formalin fixing and paraffin embedding
• Genomic abnormalities of various types are known to occur in cancer cells, and increase in their quantity, distribution, and complexity as the cancer cells are replicated over time. These genomic abnormalities include gene deletions and multiplications, some of which may be related to a specific function in humans and common to certain cancer types, while others may have no overt effect or association with disease. As a result, a reference sequence of interest may undergo deletion and/or multiplication in the genome of a cancer cell, thereby making it very difficult to determine the absolute copy number of the reference sequence.
• nucleic acids DNA and RNA
• cross linking of nucleotides to themselves or proteins, depurination of nucleotides, and fragmentation of the nucleic acids are known to occur as part of the archiving of cancer samples by the FFPE method.
• the locations and extent of these modifications are random and vary greatly across FFPE samples, due in large part to one or more factors, such as variability in the sample tissue itself, reagents used in fixation and embedding, and user preferred variations in the FFPE method at different labs. These effects also make it difficult or impossible to determine the absolute copy number of the reference sequence.
• the present disclosure relates to a method, comprising quantifying a nucleic acid sequence of interest relative to a reference nucleic acid sequence, wherein at least a first minimum number of copies of the reference nucleic acid sequence is present on each of at least a second minimum number of chromosomes of the genomic DNA of the subject; and determining a copy number of the sequence of interest from the relative quantified amplified sequence of interest.
• the method may further comprise amplifying the nucleic acid sequence of interest in a sample comprising genomic DNA of a subject; and amplifying the reference nucleic acid sequence in the sample; prior to quantifying.
• the reference sequence may have at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl-329448-341534, chrl -648129-660266, chrl-222643865-228172047, chrl- 243203764-243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl6- 90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2- 243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4- 119557144-120325498, chr4-165196360-165199636, chr5
• the reference nucleic acid sequence may have at least 80% sequence identity to at least one of GGCTGYTTGCRGTAGTWRTSTRKSWRSMRSMMRMWSRMYGSMSRCARRS RARRMARWYWSTWDVWAKKMN (SEQ ID NO:l),
• the present disclosure relates to a kit, comprising a first probe which specifically hybridizes to at least a portion of at least one reference sequence that has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl -329448-341534, chrl-648129- 660266, chrl -222643865-228172047, chrl -243203764-243215874, chrlO-38741930- 38753964, chrl 1-114010-126106, chrl6-90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2-243064480-243071940, chr20-62921559- 62933673, chr3-197950387-197962431,
• the present disclosure relates to a composition, comprising a first probe which specifically hybridizes to at least a portion of at least one reference sequence that has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl -329448-341534, chrl- 648129-660266, chrl-222643865-228172047, chrl-243203764-243215874, chrlO- 38741930-38753964, chrl 1-114010-126106, chrl 6-90239446-90251554, chrl9- 183944-196032, chr2-l 14323560-114323652, chr2-243064480-243071940, chr20- 62921559-62933673, chr3-197950387-197962431, chr
• the present disclosure relates to a system, comprising a nucleic acid amplifier configured to amplify a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject and amplify a reference sequence in the sample; a reagent reservoir containing at least a first primer configured to specifically hybridize to a first end of the at least one reference sequence, wherein the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl-329448- 341534, chrl -648129-660266, chrl-222643865-228172047, chrl-243203764- 243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl 6-90239446- 90251554, chrl9-183944-1960
• Figure 1A shows the decimal point calculated copy number of IRS2 as determined by qPCR with an RNaseP reference assay.
• Figure IB shows the rounded copy number of IRS2 as determined by qPCR with an RNaseP reference assay.
• Figure 2A shows the decimal point calculated copy number of IRS2 as determined by qPCR with a reference assay based on SEQ ID NO: 1-8.
• Figure 2B shows the rounded copy number of IRS2 as determined by qPCR with a reference assay based on SEQ ID NO: 1-8.
• Figure 3A shows the decimal point calculated copy number of IRS2 as determined by qPCR with a reference assay based on SEQ ID NO: 9- 13.
• Figure 3B shows the rounded copy number of IRS2 as determined by qPCR with a reference assay based on SEQ ID NO:9-13.
• Various embodiments of the present disclosure provide reference sequences that are relatively resistant to cancer-induced genomic abnormalities and/or FFPE- induced nucleic acid modifications. More specifically, the disclosure provides target sequences in the genome that are repeated multiple times, across many chromosomes, and demonstrate substantially normal copy number and resistance to severe modifications and fragmentation, even in cancer cells subjected to FFPE.
• the present disclosure relates to a method, comprising amplifying a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject; amplifying a reference nucleic acid sequence in the sample, wherein at least one copy of the reference nucleic acid sequence is present on each of at least ten chromosomes of the genomic DNA of the subject; quantifying the amplified sequence of interest relative to the amplified reference sequence; and determining a copy number of the sequence of interest from the relative quantified amplified sequence of interest.
• Amplifying may be performed by any technique known to the person of ordinary skill in the art. Desirably, amplifying may be performed by a technique which permits quantification of the sequence of interest relative to the reference sequence. Exemplary techniques include, but are not limited to, polymerase chain reaction (PCR) (Saiki et al. (1985) Science 230: 1350), quantitative real-time PCR (qPCR), digital PCR, ligase chain reaction (LCR) (Landegren et al. (1988) Science 241 :1077-1080), helicase-dependent amplification (HDA) (Vincent et al. (2004) EMBO rep 5(8):795-800), thermostable HDA (tHDA) (An et al.
• PCR polymerase chain reaction
• qPCR quantitative real-time PCR
• digital PCR digital PCR
• LCR ligase chain reaction
• HDA helicase-dependent amplification
• tHDA thermostable HDA
• TMA transcription mediated amplification
• NEAR nicking enzyme amplification reaction
• EXPAR exponential amplification reaction
• LAMP loop mediated isothermal amplification
• RPA recombinase polymerase amplification
• amplification is performed by TaqMan quantitative polymerase chain reaction (qPCR).
• qPCR platform assays use two genome targets together to determine the copy number of a gene or region of the genome in a test sample.
• One of the genome targets is a qPCR assay for the target of interest (TOI), and the second is a qPCR reference assay for what is assumed to be a normal, unmodified region of the genome. The two assays are run simultaneously and in parallel on the same test sample.
• the Cq values of each assay may be determined by techniques known to the person of ordinary skill in the art and/or described in more detail below, and a delta Cq between them is calculated. This calculated delta Cq may then be compared to a delta Cq that is representative of a known copy number for the TOI.
• the representative delta Cq may be a delta Cq determined from a sample known to be normal (i.e., having a copy number of 2, one copy from each of a pair of chromosomes).
• This final calculated delta delta Cq between test sample and known sample/value may then be transformed into a decimal number or an integer number representing the copy number of the gene or region of genome in the test sample.
• the challenge in cancer FFPE samples is in the ability to find a reference genome target (qPCR reference assay target) that is both normal and relatively unmodified by fixation and embedding. Additionally or alternatively, for some samples the ability to find a reference genome target may be complicated by a particular disease state, which includes but is not limited to cancer, where a potential reference genomic target may be altered by the disease state and is itself multiplied relative to its typical population.
• any nucleic acid sequence from the genomic DNA of a subject and of interest to the user of the method may be amplified and quantified according to the method.
• the sequence of interest may be at least a portion of a gene which has an association with a disease.
• the sample may be any tissue likely or possibly containing the nucleic acid sequence of interest in genomic DNA.
• the method may be used to amplify and quantify a sequence of interest from tissue suspected of being cancer tissue, including tissue which has been subjected to formalin fixing and paraffin embedding (FFPE) prior to the amplifying the sequence of interest and amplifying the reference sequence.
• the sequence of interest may be at least a portion of a gene for which there exists a correlation between the gene's copy number and the presence and/or stage of a cancer.
• any reference nucleic acid sequence known or expected to be present in the genomic DNA of the sample may be amplified.
• any particular locus of a copy of a reference nucleic acid sequence may have undergone a recombination event, an aneuploidy event, or the like.
• the number of copies of the reference sequence in the sample may differ from that expected by simple counting of the number of loci of the reference sequence in a non-diseased sample from the subject or a member of the subject's species.
• the sample comprises at least one copy of the reference nucleic acid sequence on each of at least ten chromosomes of the genomic DNA of the subject.
• the presence of multiple, physically dispersed copies of the reference sequence may smooth or average out the effects of individual disruptions or duplications of various loci.
• the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl- 329448-341534, chrl -648129-660266, chrl-222643865-228172047, chrl- 243203764-243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl6- 90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2- 243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431 , chr4- 119557144-120325498, chr4-165196360-165199636, chr5
• the first minimum number may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 copies.
• the second minimum number may be 5, 6, 7, 8, 9, 10, or 11 chromosomes.
• the first and second minimum numbers may be enumerated, estimated, or predicted based on any available human reference genome.
• the reference sequence has at least 80% sequence identity to at least one of
• the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO: 1-8.
• the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO:9-13.
• a first set of sequences correspond to sequences found in the human genome at chrl : 121836-121905, chrl :243203810-243203879, chrl :341419+341488, chrl :648175-648244, chr2:243071825+243071894, chr3 : 197962362+197962431 , chr4: 119569113+ 119569182, chr5: 180768034+180768103, chr6: 132997-133066, chr6: 170922434+170922503, chrl0:38753924+38753993, chrl 1 : 114056-114125, chrl6:90251439+90251508, chrl9: 183990-184059, chr20:62933558+62933627, chrUn_gl000227:
• a second set of sequences, SEQ ID NO:9-13, correspond to sequences found in the human genome at chrl :224126101-224126167, chrl :228152189+228152255, chrl :243203891-243203957, chrl :341341+341407, chrl :648256-648322, chr2:243071747+243071813, chr3: 197962284+ 197962350, chr4: 119569035+119569101 , chr5: 180767956+180768022, chr6: 133078-133144, chr6: 170922356+170922422, chr8: 143260-143326, chrl0:38753846+38753912, chrl l : l 14137-114203, chrl6:90251361+90251427, chrl9
• each of the first set and the second set of sequences are both highly repeated (-20 copies in the human genome), physically dispersed throughout the human genome, and relatively more resistant to disruption and/or duplication by FFPE than typical genomic DNA sequences.
• a sequence having at least 80% identity to one or more of SEQ ID NO: 1-13 may be particularly suitable as a reference sequence, especially in samples suspected of being cancer tissue, particular FFPE-processed tissue.
• the amplifying steps yield an amplified sequence of interest and an amplified reference sequence.
• the amplifying steps yield an amplified sequence of interest and an amplified reference sequence.
• the relative amounts of the amplified sequence of interest and the amplified reference sequence will be proportional to their copy number in the genomic DNA of the sample.
• the method may comprise quantifying the amplified sequence of interest relative to the amplified reference sequence.
• the quantifying may be performed by any technique known to the person of ordinary skill in the art. For example, by the use of two probes, each comprising a fluorescent moiety at a first end and a quencher for that fluorescent moiety at a second end, with one probe specifically hybridizing to the sequence of interest and the other specifically hybridizing to the reference sequence, in TaqMan qPCR, cleavage of the quencher by the action of Taq polymerase will generate a fluorescence signal proportional to the amount of probe hybridized to the sequence of interest or the reference sequence.
• the relative quantity of the amplified sequence of interest would be 0.2. (As will be apparent to the person of ordinary skill in the art, alternative mathematically equivalent expressions may be used to arrive at a relative quantity).
• amplification of the TOI and the reference sequence may be omitted.
• Techniques for quantifying non-amplified nucleic acid sequences are known to the person of ordinary skill in the art.
• fold change refers to the amount of amplified product (which relates to the copy number) in the sequence of interest relative to that of the reference genome target.
• Fold change can be quantified using any of several available methods, including but not limited to those described by Livak, et al. (Methods, 25:402-408 (2001)), commercially available products such as CopyCallerTM (Applied Biosystems), or any other suitable algorithm for comparing amounts of fluorescence signals.
• fold change is determined by comparing the CT of the sequence of interest to the CT of the reference genome target.
• Some suitable algorithms include but are not limited to, the methods described in U.S. Application Serial No. 13/107786, "Karyotyping Assay” filed on May 13, 2011, the disclosure of which is hereby incorporated by reference in its entirety.
• the quantifying step yields a relative quantified amplified sequence of interest.
• the method may then comprise determining a copy number of the sequence of interest from the relative quantified amplified sequence of interest. Determining requires an indication of the copy number of the reference sequence. Such an indication may be provided by analysis of the genome of a non-diseased sample from the subject or one or more members of the subject's species. This technique may be especially suitable, regarding samples suspected of being cancer tissue, for a reference sequence that is one or more of highly repeated, physically dispersed, and relatively resistant to disruption and/or duplication by FFPE.
• the copy number of a reference sequence having at least 80% identity to one or more of SEQ ID NO: 1-13 in a non-diseased sample may be expected to be substantially equal to the copy number of the reference sequence in a sample suspected of being cancer tissue.
• the copy number of the reference sequence is 20
• this is a simple probe-based example of a copy number calculation. It is a routine matter for the person of ordinary skill in the art, having the benefit of the present disclosure, to perform copy number calculation for other assay techniques, such as qPCR.
• the determined copy number of the sequence of interest may be used for any purpose which would commend itself to the person of ordinary skill in the art.
• the method may further comprise diagnosing the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.
• the present disclosure relates to a kit comprising a first probe which specifically hybridizes to at least a portion of at least one reference sequence having at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl -329448-341534, chrl-648129- 660266, chrl -222643865-228172047, chrl -243203764-243215874, chrlO-38741930- 38753964, chrl 1-114010-126106, chrl6-90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2-243064480-243071940, chr20-62921559- 62933673, chr3-197950387-197962431, chrr
• the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO: 1-31.
• the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• a "probe,” as used herein, refers to a compound comprising a nucleic acid sequence and a detectable moiety. As such, and for the avoidance of doubt, any “probe” referred to herein is non-naturally occurring.
• the first probe comprises a nucleic acid sequence configured to specifically hybridize to at least the portion of the at least one reference sequence, a fluorescent reporter at a first end of the nucleic acid sequence, and a fluorescent quencher at a second end of the nucleic acid sequence.
• the nucleic acid sequence is configured to specifically hybridize to the entirety of at least one reference sequence.
• a percentage of sequence identity can be determined by any technique known to the person of ordinary skill in the art.
• the reference sequence has at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%,
• the first probe may allow the detection of at least the portion of the at least one reference sequence.
• the kit may comprise other components.
• the kit may further comprise a first primer configured to specifically hybridize to a first end of the at least one reference sequence, and a second primer configured to specifically hybridize to a sequence complementary to a second end of the at least one reference sequence.
• primers nucleic acid molecules which, in the presence of the at least one reference sequence and other reagent(s), may allow amplification of the at least one reference sequence.
• the kit may further comprise a second probe which specifically hybridizes to at least a portion of at least one nucleic acid sequence of interest.
• the second probe may have the same characteristics as the first probe described above.
• any nucleic acid sequence of interest may be the hybridization target of the second probe.
• the sequence of interest is a portion or the entirety of a gene associated with a cancer.
• the kit may further comprise a third primer configured to specifically hybridize to a first end of the at least one nucleic acid sequence of interest, and a fourth primer configured to specifically hybridize to a sequence complementary to a second end of the at least one nucleic acid sequence of interest.
• the present disclosure relates to a composition, comprising a first probe which specifically hybridizes to at least a portion of at least one reference sequence having at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl -329448-341534, chrl- 648129-660266, chrl-222643865-228172047, chrl-243203764-243215874, chrlO- 38741930-38753964, chrl 1-114010-126106, chrl 6-90239446-90251554, chrl9- 183944-196032, chr2-l 14323560-114323652, chr2-243064480-243071940, chr20- 62921559-62933673, chr3-197950387-197962431, chr4-l 195571
• the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO: 1-31.
• the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• the first probe may be substantially the same as the first probe of the kit, described above.
• the composition may further comprise one or more of (i) a first primer configured to specifically hybridize to a first end of at least one reference sequence, and a second primer configured to specifically hybridize to a sequence complementary to a second end of at least one reference sequence; (ii) a second probe which specifically hybridizes to at least a portion of at least one nucleic acid sequence of interest; or (iii) a third primer configured to specifically hybridize to a first end of at least one nucleic acid sequence of interest, and a fourth primer configured to specifically hybridize to a sequence complementary to a second end of at least one nucleic acid sequence of interest, substantially the same as the corresponding further component(s) of the kit, described above.
• the present disclosure relates to a system, comprising:
• a nucleic acid amplifier configured to amplify a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject and amplify a reference sequence in the sample;
• a reagent reservoir containing at least a first primer configured to specifically hybridize to a first end of at least one reference sequence, wherein the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl-329448-341534, chrl -648129-660266, chrl- 222643865-228172047, chrl -243203764-243215874, chrlO-38741930-38753964, chrl l-114010-126106, chrl6-90239446-90251554, chrl9-183944-196032, chr2- 114323560-114323652, chr2-243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4-
• a detector configured to provide a first indication relating to an amount of the amplified sequence of interest and a second indication relating to an amount of the amplified reference sequence
• a controller configured to quantify the amplified sequence of interest relative to the amplified reference sequence, based at least in part on the first indication and the second indication; and determine a copy number of the sequence of interest from the relative quantified amplified sequence of interest.
• Nucleic acid amplifiers are known to the person of ordinary skill in the art. Generally, nucleic acid amplifiers use one or more primers and one or more chemical or enzymatic agents to copy a template nucleic acid sequence, such as a sequence of interest or a reference sequence. Such copying can be cycled multiple times to yield relatively large amounts of the sequence of interest and the reference sequence. Desirably, the nucleic acid amplifier is configured to amplify the sequence of interest and the reference sequence simultaneously and in parallel, e.g., by adding different sets of primers, one specific to the sequence of interest and the other specific to the reference sequence, to otherwise identical reaction solutions, such as in different wells of a multi-well plate.
• the system also comprises a reagent reservoir.
• the reagent reservoir contains materials required for the amplification reaction to occur, such as primers, chemical or enzymatic agents, free nucleotides incorporable into copies of template sequences, etc.
• the reagent reservoir may also contain one or more probes or other compounds comprising detectable moieties. Any of these materials may be stored separately and/or two or more thereof may be combined for storage in the reagent reservoir. These materials are generally in aqueous solution and can be introduced to reaction solution(s) by techniques known to the person of ordinary skill in the art. Such introduction can occur once or multiple times before, during, or after an amplification process. For example, some reagent(s) may be added once per amplification cycle.
• the reagent reservoir containing at least a first primer configured to specifically hybridize to a first end of the at least one reference sequence, wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO: l-31 , such as SEQ ID NO: l-13, and a second primer configured to specifically hybridize to a sequence complementary to a second end of the at least one reference sequence.
• the system also comprises a detector.
• the detector may be configured to detect a probe for the sequence of interest, the reference sequence, or both. Upon detection, the detector may perform various signal processing and/or analysis operations to provide a first indication relating to an amount of the amplified sequence of interest and a second indication relating to an amount of the amplified reference sequence.
• the system also comprises a controller.
• the controller may be configured to quantify the amplified sequence of interest relative to the amplified reference sequence, based at least in part on the first indication and the second indication; and determine a copy number of the sequence of interest from the relative quantified amplified sequence of interest. It may store the determined copy number in a memory, display it to a user, write it to a computer-readable file, or the like.
• the controller may be configured to diagnose the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.
• nucleic acid amplifier the reagent reservoir, the detector, and the controller have been described separately above, any two or more thereof may be components of a single apparatus.
• the disclosure provides:
• a method comprising: amplifying a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject; amplifying a reference sequence in the sample, wherein the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl-329448- 341534, chrl -648129-660266, chrl -222643865-228172047, chrl -243203764- 243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl 6-90239446- 90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2-243064480- 243071940, chr20-62921559-62933673, chr3-197950387-
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NOs: 1-31. 3. In the method, the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• the sample can include tissue suspected of being cancer tissue.
• the sample has been subjected to formalin fixing and paraffin embedding (FFPE) prior to amplifying the sequence of interest and amplifying the reference sequence.
• FFPE formalin fixing and paraffin embedding
• the method can also include: diagnosing the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.
• amplifying the sequence of interest and amplifying the reference sequence can be performed by TaqMan quantitative polymerase chain reaction (qPCR).
• a kit comprising:a first probe which specifically hybridizes to at least a portion of at least one reference sequence having at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl- 329448-341534, chrl -648129-660266, chrl-222643865-228172047, chrl- 243203764-243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl6- 90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2- 243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4- 119557144-120325498, chrr
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• the first probe can include a nucleic acid sequence configured to specifically hybridize to at least the portion of the at least one reference sequence, a fluorescent reporter at a first end of the nucleic acid sequence, and a fluorescent quencher at a second end of the nucleic acid sequence.
• the kit can further include: a first primer configured to specifically hybridize to a first end of the at least one reference sequence, and a second primer configured to specifically hybridize to a sequence complementary to a second end of the at least one reference sequence.
• the kit can further include: a second probe which specifically hybridizes to at least a portion of at least one nucleic acid sequence of interest.
• the kit can further include: a third primer configured to specifically hybridize to a first end of the at least one nucleic acid sequence of interest, and a fourth primer configured to specifically hybridize to a sequence complementary to a second end of the at least one nucleic acid sequence of interest.
• a composition that includes: a first probe which specifically hybridizes to at least a portion of at least one reference sequence having at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl-329448-341534, chrl -648129-660266, chrl-222643865-228172047, chrl- 243203764-243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl6- 90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114323652, chr2- 243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4- 119557144-120325498, chr
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-31.
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• the first probe can include a nucleic acid sequence configured to specifically hybridize to at least the portion of the at least one reference sequence, a fluorescent reporter at a first end of the nucleic acid sequence, and a fluorescent quencher at a second end of the nucleic acid sequence.
• the composition can further include: a first primer configured to specifically hybridize to a first end of the at least one reference sequence, and a second primer configured to specifically hybridize to a sequence complementary to a second end of the at least one reference sequence.
• composition can further include: a second probe which specifically hybridizes to at least a portion of at least one nucleic acid sequence of interest.
• the composition can further include: a third primer configured to specifically hybridize to a first end of the at least one nucleic acid sequence of interest, and a fourth primer configured to specifically hybridize to a sequence complementary to a second end of the at least one nucleic acid sequence of interest.
• a system that includes: a nucleic acid amplifier configured to amplify a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject and amplify a reference sequence in the sample, a reagent reservoir containing at least a first primer configured to specifically hybridize to a first end of the at least one reference sequence, wherein the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chrl-121790-133586, chrl-329448-341534, chrl -648129-660266, chrl-222643865-228172047, chrl- 243203764-243215874, chrlO-38741930-38753964, chrl 1-114010-126106, chrl6- 90239446-90251554, chrl9-183944-196032, chr2-l 14323560-114
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-31.
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• the sample can include tissue suspected of being cancer tissue.
• the sample has been subjected to formalin fixing and paraffin embedding (FFPE).
• FFPE formalin fixing and paraffin embedding
• the controller is further configured to indicate the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.
• the nucleic acid amplifier is configured to amplify the sequence of interest and amplify the reference sequence by TaqMan quantitative polymerase chain reaction (qPCR).
• qPCR TaqMan quantitative polymerase chain reaction
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-38.
• the reference sequence can have at least 80% sequence identity to at least one of SEQ ID NO: 1-13.
• the sample includes tissue suspected of being cancer tissue. 33.
• the sample has been subjected to formalin fixing and paraffin embedding (FFPE) prior to amplifying the sequence of interest and amplifying the reference sequence.
• FFPE formalin fixing and paraffin embedding
• the method can further include: diagnosing the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.
• SEQ ID NO: l-13 were identified by a two-stage process.
• a bioinformatics algorithm was used to identify candidate targets in the genome that met certain criteria associated with substantially normal copy number, even in cancer cells subjected to FFPE.
• sequences suspected of being relatively resistant to copy number abnormalities in cancer cells and/or cells subjected to FFPE were used to query publicly- available human genomic databases, and only those sequences returning multiple hits were considered as candidate targets for further testing.
• SEQ ID NO: l-31 The first stage identified SEQ ID NO: l-31.
• SEQ ID NO:2-8 and 10-13 are located in the genome at the loci given supra.
• SEQ ID NO: 14-31 are located in the genome at least at the following loci:
• the first multicopy reference assay corresponding to SEQ ID NO: 1-8, used a first set comprising forward primer, reverse primer, and probe sequence.
• the second multicopy reference assay corresponding to SEQ ID NO:9-13, used a second set comprising forward primer, reverse primer, and probe sequence.
• compositions, methods, and/or systems disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods, and/or systems and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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